WO2019103456A2 - Biomarker composition for diagnosing radiation-resistant cancer or for predicting prognosis of radiation therapy containing pmvk as active ingredient - Google Patents

Biomarker composition for diagnosing radiation-resistant cancer or for predicting prognosis of radiation therapy containing pmvk as active ingredient Download PDF

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WO2019103456A2
WO2019103456A2 PCT/KR2018/014363 KR2018014363W WO2019103456A2 WO 2019103456 A2 WO2019103456 A2 WO 2019103456A2 KR 2018014363 W KR2018014363 W KR 2018014363W WO 2019103456 A2 WO2019103456 A2 WO 2019103456A2
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Prior art keywords
pmvk
cancer
radiation
protein
expression level
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PCT/KR2018/014363
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French (fr)
Korean (ko)
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WO2019103456A3 (en
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최은경
정성윤
송시열
박윤용
박석순
주은진
고은정
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울산대학교 산학협력단
재단법인 아산사회복지재단
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Priority claimed from KR1020180143493A external-priority patent/KR102141997B1/en
Application filed by 울산대학교 산학협력단, 재단법인 아산사회복지재단 filed Critical 울산대학교 산학협력단
Priority to JP2020546259A priority Critical patent/JP6924907B2/en
Priority to EP18881259.8A priority patent/EP3715475A4/en
Priority to CN201880087275.XA priority patent/CN111630185A/en
Priority to US16/766,297 priority patent/US11674184B2/en
Publication of WO2019103456A2 publication Critical patent/WO2019103456A2/en
Publication of WO2019103456A3 publication Critical patent/WO2019103456A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a biomarker composition for diagnosing radiation-resistant cancer comprising phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient, and a method for diagnosing radiation-resistant cancer using the same. Further, the present invention relates to a biomarker composition for predicting the prognosis of radiation therapy for cancer patients including PMVK as an active ingredient, and a method for predicting the prognosis of radiation therapy for cancer patients using the composition.
  • PMVK phosphomevalonate kinase
  • Cancer treatment methods include surgical operations, chemical medication, and radiotherapy. Radiation therapy is becoming more important in recent years. In cases where surgical treatment is difficult, radiation therapy alone has been effective in curing tumors. Radiation therapy has also been developed over the years, and radiation therapy has been applied to patients with no special pain or discomfort. As a way to treat it effectively.
  • Radiation therapy sensitizers reported to date are mainly anticancer drugs, such as taxol and cisplatin, for breast, uterine, and cervical cancer. It has been reported that it can be used as a radiotherapy sensitizer in solid tumors such as lung cancer, stomach cancer and colon cancer.
  • these radiation-sensitive sensitizers are substances that are used as anticancer drugs themselves, and thus exhibit high side effects, and thus their use is limited.
  • Another object of the present invention is to provide a biomarker composition for predicting the prognosis of radiation therapy for a cancer patient comprising PMVK as an active ingredient, a composition for predicting the prognosis of radiation therapy for cancer patients, which comprises a preparation capable of measuring the expression level of PMVK .
  • Another object of the present invention is to provide a method for diagnosing radiation-resistant cancer using PMVK protein expression and activity level and a method for predicting the prognosis of radiation therapy for cancer patients.
  • the present invention provides a biomarker composition for diagnosing radiation-resistant cancer, comprising as an active ingredient a phosphomevalonate kinase (PMVK) protein or a gene encoding the same.
  • a biomarker composition for diagnosing radiation-resistant cancer comprising as an active ingredient a phosphomevalonate kinase (PMVK) protein or a gene encoding the same.
  • PMVK phosphomevalonate kinase
  • the present invention also provides a composition for the diagnosis of radiation-resistant cancer, which comprises a preparation capable of measuring the expression level of PMVK as an active ingredient, and a radiation-resistant cancer diagnostic kit comprising the same.
  • the present invention provides a biomarker composition for predicting the radiation therapy prognosis of a cancer patient, which comprises a phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient.
  • a biomarker composition for predicting the radiation therapy prognosis of a cancer patient which comprises a phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient.
  • PMVK phosphomevalonate kinase
  • the present invention also provides a composition for predicting the prognosis of radiation therapy for cancer patients, which comprises a preparation capable of measuring the expression level of PMVK, and a kit for predicting the prognosis of radiation therapy for cancer patients containing the composition.
  • a pharmaceutical composition for promoting radiation sensitivity to a cancer cell comprising, as an active ingredient, a phosphomevalonate kinase (PMVK) protein expression or activity inhibitor.
  • PMVK phosphomevalonate kinase
  • a method for detecting cancer comprising the steps of: (1) contacting a test substance with a cancer cell; (2) measuring the expression or activity level of PMVK protein in the cancer cells in contact with the test substance; And (3) selecting a test substance whose expression or activity level of the PMVK protein is decreased as compared with a control sample.
  • the present invention also provides a method for screening a radiation sensitivity enhancer for cancer cells.
  • the present invention also provides the use of the PMVK protein or a gene encoding the same as a biomarker for the diagnosis of radiation-resistant cancer.
  • the present invention also provides the use of an agent capable of measuring the level of expression of PMVK for the diagnosis of radiation resistant cancer.
  • the present invention provides a biomarker for predicting the prognosis of radiation therapy in a cancer patient, which uses the PMVK protein or a gene encoding the PMVK protein.
  • the present invention also provides the use of a pharmaceutical agent capable of measuring the level of expression of PMVK for predicting the prognosis of radiation therapy in cancer patients.
  • the present invention also provides the use of a PMVK protein expression or activity inhibitor for the manufacture of a medicament for enhancing radiation sensitivity to cancer cells.
  • the present invention relates to a biomarker composition for diagnosing radiation-resistant cancer or a biomarker composition for predicting the prognosis of radiation therapy, which comprises a phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient,
  • PMVK phosphomevalonate kinase
  • the PMVK gene which can enhance the sensitivity of radiation in A549 cells, was identified, and A549 cells and Miapaca-2 cells of pancreatic cancer cells were constructed using shRNA, And PMVK overexpression contributes to the resistance of radiation through clinical data analysis.
  • PMVK can be used as a therapeutic target gene to overcome radiation resistance in human lung and pancreatic cancer. Therefore, PMVK Genes that increase radiation sensitivity to targets Developed ryoje or other substances can be further increased the therapeutic effect of radiation.
  • FIG. 1 shows the result of confirming the radiation sensitivity-enhancing gene PMVK through human kinase siRNA library screening in one embodiment of the present invention.
  • A A549 human lung cancer cell line was screened by human kinase siRNA library to determine the radiation sensitivity promoting gene PMVK
  • B PMVK siRNA inhibited the expression of PMVK protein and the result of reduction of cell viability was confirmed by radiation treatment).
  • FIG. 2 shows the result of performing colonization formation survival analysis by constructing a PMVK shRNA stable cell line and treating the established cells with radiation, in one embodiment of the present invention.
  • A Expression of PMVK protein in A549 / shPMVK stable cell line
  • D The inhibitory effects of D: Miapaca-2 / shPMVK on the inhibition of PMVK protein expression in C: Miapaca-2 / shPMVK stable cell line were investigated as a result of confirming the effect of radiation on colony formation in
  • B A549 / shPMVK stable cell line.
  • the effect of radiation on colony formation was confirmed in shPMVK stable cell lines.
  • Figure 3 shows the results of an evaluation of efficacy against radiation in an A549 / shPMVK cell transplantation mouse model in one embodiment of the present invention (A: volume growth of cancer compared to volume B: weight of mouse , C: comparison of the size of the extracted cancer, and D: comparison of the weight of the extracted cancer).
  • Figure 4 shows the results of clinical genomic data analysis according to the expression of PMVK in radiation treatment of lung cancer patients in one embodiment of the present invention.
  • Figure 5 shows the results of an efficacy assessment for radiation in a Miapaca-2 / shPMVK cell transplant mouse model in one embodiment of the invention.
  • the present invention provides a biomarker composition for diagnosing radiation-resistant cancer, which comprises as an active ingredient a phosphomevalonate kinase (PMVK) protein or a gene coding therefor.
  • a biomarker composition for diagnosing radiation-resistant cancer which comprises as an active ingredient a phosphomevalonate kinase (PMVK) protein or a gene coding therefor.
  • PMVK phosphomevalonate kinase
  • the biomarker composition further comprises a known radiation resistance biomarker, and known radiation resistance biomarkers include, but are not limited to, CD133, CD144, and CD24.
  • diagnosing herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of the object, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).
  • the present invention provides a radiation-resistant cancer diagnostic composition
  • a radiation-resistant cancer diagnostic composition comprising as an active ingredient a preparation capable of measuring the expression level of PMVK.
  • the agent capable of measuring the expression level of PMVK may be a primer or a probe specifically binding to the PMVK gene, an antibody, a peptide, an aptamer or a compound specifically binding to the PMVK protein, But is not limited thereto.
  • the cancer may be lung cancer or pancreatic cancer, but is not limited thereto.
  • the present invention also provides a radiation-resistant cancer diagnostic kit comprising the composition.
  • the present invention provides a biomarker composition for predicting the radiotherapeutic prognosis of a cancer patient comprising a phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient.
  • a biomarker composition for predicting the radiotherapeutic prognosis of a cancer patient comprising a phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient.
  • PMVK phosphomevalonate kinase
  • prognostic prediction refers to an act of predicting the course and outcome of a disease. More specifically, the prognosis prediction means that the progress of the disease after the treatment depends on the physiological or environmental condition of the patient, and it means all actions that predict the course of the disease after treatment considering the condition of the patient in a comprehensive manner .
  • the prognosis prediction can be interpreted as predicting the disease-free survival rate or the survival rate of the cancer patient by anticipating the course of the disease and the cure after the radiation therapy of the cancer patient. For example, predicting a "good prognosis" indicates a high level of disease-free survival or survival rate in cancer patients after radiation therapy, which implies that cancer patients are more likely to be treated, and the prediction of "poor prognosis"
  • the disease-free survival rate or survival rate of cancer patients after radiation therapy is low, meaning that cancer is likely to recur from cancer patients or die from cancer.
  • the present invention also provides a composition for predicting the prognosis of radiation therapy for a cancer patient, which comprises a preparation capable of measuring the expression level of PMVK as an active ingredient.
  • the agent capable of measuring the expression level of PMVK may be a primer or a probe specifically binding to the PMVK gene, an antibody, a peptide, an aptamer or a compound specifically binding to the PMVK protein.
  • the cancer may be lung cancer or pancreatic cancer, but is not limited thereto.
  • the present invention provides a kit for predicting the prognosis of radiation therapy for a cancer patient comprising the composition.
  • primer refers to a nucleic acid sequence capable of forming base pairs with a complementary template with a short free 3'-hydroxyl group and having a short Refers to a nucleic acid sequence.
  • Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures.
  • reagents for polymerization i. E., DNA polymerase or reverse transcriptase
  • the PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.
  • the term " probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to an mRNA. The presence or absence of a specific mRNA, Can be confirmed.
  • the probe can be produced in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art.
  • the term " antibody" means a specific immunoglobulin as indicated in the art and directed against an antigenic site.
  • An antibody in the present invention refers to an antibody that specifically binds to the PMVK of the present invention, and the antibody can be produced according to a conventional method in the art.
  • the forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies.
  • the antibody refers to a complete form having two full-length light chains and two full-length heavy chains.
  • the antibody also includes a special antibody such as a humanized antibody.
  • the term " peptide" has a high binding capacity to the target material and does not cause denaturation during thermal / chemical treatment.
  • the molecular size is small, it can be used as a fusion protein by attaching to other proteins. It can be used as a diagnostic kit and a drug delivery material because it can be specifically attached to a polymer protein chain.
  • aptamer refers to a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid having a stable tertiary structure by itself and having a characteristic capable of binding with high affinity and specificity to a target molecule ). ≪ / RTI > As described above, since the aptamer is composed of a polynucleotide which is capable of specifically binding to an antigenic substance like the antibody and is more stable than the protein, has a simple structure, and is easy to synthesize, .
  • the kit of the present invention comprises an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a labeling substance that is colored by the reaction with the substrate, a coloring substrate solution that reacts with the labeling substance, Enzyme reaction termination solutions, and the like, and can be made from a number of separate packaging or compartments, including the reagent components used.
  • the present invention provides a pharmaceutical composition for promoting radiation sensitivity to a cancer cell comprising, as an active ingredient, a phosphomevalonate kinase (PMVK) protein expression or activity inhibitor.
  • a pharmaceutical composition for promoting radiation sensitivity to a cancer cell comprising, as an active ingredient, a phosphomevalonate kinase (PMVK) protein expression or activity inhibitor.
  • PMVK phosphomevalonate kinase
  • the PMVK expression inhibitor may be any one selected from the group consisting of an antisense nucleotide complementarily binding to the mRNA of the PMVK gene, a small interfering RNA (siRNA) and a short hairpin RNA (shRNA)
  • the PMVK activity inhibitor may be any one selected from the group consisting of low molecular compounds, peptides, peptide mimetics, aptamers, antibodies and natural substances that specifically bind to the PMVK protein, but the present invention is not limited thereto.
  • the cancer cells may be lung cancer cells or pancreatic cancer cells.
  • the pharmaceutical composition of the present invention may contain a chemical substance, a nucleotide, an antisense, an siRNA oligonucleotide and a natural product extract as an active ingredient.
  • the pharmaceutical composition or combination preparation of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients.
  • the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants , A lubricant or a flavoring agent may be used.
  • the pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration.
  • Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, suspending agents or injectable solutions or suspensions .
  • the pharmaceutical compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, ≪ / RTI >
  • An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of the disease.
  • the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like.
  • the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg for a compound once to several times a day, , 0.1 ng / kg to 10 g / kg for a protein or an antibody, 0.01 ng / kg to 10 g / kg for an antisense nucleotide, siRNA, shRNAi or miRNA.
  • the present invention provides a method for detecting cancer, comprising the steps of: (1) measuring the mRNA expression level or the PMVK protein expression level of a PMVK gene from a sample isolated from a cancer patient; (2) comparing the mRNA expression level of the PMVK gene or the expression level of the PMVK protein with a control sample; And (3) determining that the mRNA expression level of the PMVK gene or the expression level of the PMVK protein is higher than that of the control sample, a step of determining that the cancer is a radiation-resistant cancer.
  • the cancer may be lung cancer or pancreatic cancer, but is not limited thereto.
  • a sample isolated from a cancer patient refers to a level of expression of PMVK gene or PMVK protein, which is a biomarker for the diagnosis of radiation-resistant cancer, in tissues, cells, whole blood, serum, plasma, saliva, , A cerebrospinal fluid, or a sample such as urine.
  • the method for measuring the mRNA expression level may be RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) ), Northern blotting and DNA chips, but are not limited thereto.
  • the method for measuring the protein expression level may be Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, But are not limited to, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, Complement Fixation Assays, FACS and protein chips.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Ouchterlony immunodiffusion but are not limited to, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, Complement Fixation Assays, FACS and protein chips.
  • radiation-resistant cancer diagnosis is made to confirm whether cancer cells are resistant to radiation or sensitive to radiation therapy strategies and radiation therapy effects of cancer patients.
  • the present invention relates to (1) contacting a test substance to a cancer cell; (2) measuring the expression or activity level of PMVK protein in the cancer cells in contact with the test substance; And (3) selecting a test substance whose expression or activity level of the PMVK protein is decreased as compared with a control sample.
  • the present invention also provides a method for screening a radiation sensitivity enhancer for cancer cells.
  • test substance used in reference to the screening method of the present invention refers to an unknown candidate substance used in screening in order to examine whether it affects the expression amount of a gene or affects the expression or activity of a protein. do.
  • samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.
  • the present invention also provides the use of the PMVK protein or a gene encoding the same as a biomarker for the diagnosis of radiation-resistant cancer.
  • the present invention also provides the use of an agent capable of measuring the level of expression of PMVK for the diagnosis of radiation resistant cancer.
  • the present invention provides a biomarker for predicting the prognosis of radiation therapy in a cancer patient, which uses the PMVK protein or a gene encoding the PMVK protein.
  • the present invention also provides the use of a pharmaceutical agent capable of measuring the level of expression of PMVK for predicting the prognosis of radiation therapy in cancer patients.
  • the present invention also provides the use of a PMVK protein expression or activity inhibitor for the manufacture of a medicament for enhancing radiation sensitivity to cancer cells.
  • A549 human lung cancer cells purchased from ATCC were subjected to a three-step screening of genes that enhance sensitivity to radiation using the Dharmacon siGENOME ® SMARTpool ® siRNA library (718 human protein kinase: G-003505) .
  • siRNA is Lipofectamine (Lipofectamine ®) RNAiMAX reagent (Reagent) in a 96-well plate, each present in a 50 nM concentration was added to 0.5 ⁇ l (Invitrogen 13778-150), A549 cells seeded 0.3 ⁇ 10 4 pieces And cultured for 24 hours. The cultured cells were treated with 8 Gy of radiation using a 6-MV photon beam linear accelerator. On the 9th day after radiation treatment, cell viability was analyzed three times through the CCK-8 assay. As a result, information was obtained that the PMVK gene is a candidate gene related to the sensitivity to radiation.
  • the siRNA library siGENOME ® SMARTpool ® human PMVK siRNA (Target Sequences: UUUAUCCGCUCCAGACUUU (SEQ ID NO: 1), CGAGAACCAUGGAGUUGAA (SEQ ID NO: 2), AAUGUGGCCUGGACAACUU (SEQ ID NO: 3), GGUGAGUGACACACGGAGA
  • the knock-down of the protein by Cat # M-006782-01 was determined by incubating the 24-well plate in which PMVK siRNA was present at a concentration of 50 nM with Lipofectamine (Lipofectamine ®) RNAiMAX reagent (Reagent) (Invitrogen: 13778-150) for 1 ⁇ l added and the the A549 cell culture 24 hours after a 3 ⁇ 10 seeded into four, and then extracting the protein from one well behind using antibodies PMVK Western blotting. The remaining 24 wells were irradiated with 5 Gy and 10 Gy, and CCK-8
  • siScramble control group, Target Sequences: UGGUUUACAUGUCGACUAA (SEQ ID NO: 5), UGGUUUACAUGUUGUGUGA (SEQ ID NO: 6 ), UGGUUUACAUGUUUUCUGA (SEQ ID NO: 7), UGGUUUACAUGUUUCCUA (SEQ ID NO: 8), Damacon, Non-Targeting pool, Cat # D-001810-10-20)
  • FIG. 1B it was confirmed by Western blotting that the protein was knocked down by PMVK siRNA, and it was once again confirmed that the survival rate of the cells decreased during the radiation treatment.
  • PMVK shRNA stable cell lines were constructed on A549 (lung cancer) and Miapaca-2 (pancreatic cancer; purchased from ATCC) cells for stable knockdown of PMVK.
  • the damask cone PMVK shRNA (Clone ID: V3LHS_350080, mature antisense (Mature Antisense): TCTGACTCAGCATCGTCCA; SEQ ID NO: 9) (Roche FuGENE ® transfection reagent (Tansfection Reagent) to A549 cells and Miapaca-2 cells: REF 04 (Two clones: A549 / shPMVK # 1 and A549 / shPMVK # 3) and Miapaca-2 / shPMVK # 3 were selected for 15 days with puromycin 4 ⁇ g / ml for transfection,
  • the shPMVK cell line (3 clones; Mia / shPMVK # 1, Mia / shPMVK # 2, Mia / shPMVK # 3)
  • A549 / shPMVK stable cell line and Miapaca-2 / shPMVK stable cell line enhance the sensitivity of radiation
  • two cell lines were treated with radiation and then subjected to a colony forming assay Respectively.
  • the cells were treated with 0, 2, 4, 5, 8, and 10 Gy of radiation after seeding 200 or 2000 cells in a 6-well plate. After 7 to 14 days, the cells were treated with 20% methanol and 0.5% And quantitatively analyzed by counting the number of staining.
  • A549 / A549 / NS, A549 / shPMVK # 1 (stable A549 cell line transfected with shRNA of SEQ ID NO: 9) and A549 / shPMVK # 3 (SEQ ID NO: 9) in the nude right calf for in vivo experiments the shRNA in a stable 1 ⁇ 10 6 A549 cell line) cells transfected dogs were each implanted to form a cancerous tissue. After irradiation, 5 Gy of radiation was irradiated twice at intervals of two days, and the size was measured using a caliper three times a week. The change of cancer growth was observed for 30 days. At the same time, the mouse body weight was also measured. Size and weight. At this time, the arm size was calculated as shown in the following formula (1).
  • Tumor volume [length ⁇ width 2 ] ⁇ 0.5
  • the survival rate of patients with high PMVK expression was poor in the radiotherapy patient group, while the survival rate of patients with low PMVK expression was improved by radiotherapy.
  • the survival rate was not affected regardless of the presence or absence of PMVK in patients without radiation therapy.
  • the radiation 2 Gy was irradiated three times at intervals of two days, and the size was measured using a caliper three times a week to observe the change of cancer growth for 30 days. At the same time, mouse weight was also measured and measured. At this time, the arm size was calculated as shown in the following formula (1).
  • Tumor volume [length ⁇ width 2 ] ⁇ 0.5

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Abstract

The present invention relates to a biomarker composition for diagnosing radiation-resistant cancer containing PMVK as an active ingredient, and a method for diagnosing radiation-resistant cancer using the same, wherein when the PMVK was knocked down, it was confirmed that the survival rate of cancer cells decreases in the radiation treatment, and on the basis of this, it was suggested that it is possible to use the PMVK as a radiation therapy resistance-related factor for cancer, whereby the PMVK can be utilized as a novel target that can enhance the radiation therapy effect on human cancer cells.

Description

PMVK를 유효성분으로 포함하는 방사선 저항성 암 진단용 또는 방사선 치료 예후 예측용 바이오마커 조성물Biomarker composition for diagnosing radiation-resistant cancer or for predicting the prognosis of radiation therapy containing PMVK as an active ingredient
본 발명은 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 방사선 저항성 암 진단용 바이오마커 조성물 및 이를 이용한 방사선 저항성 암 진단 방법에 관한 것이다. 또한, PMVK를 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 바이오마커 조성물 및 이를 이용한 암 환자의 방사선 치료 예후 예측 방법에 관한 것이다.The present invention relates to a biomarker composition for diagnosing radiation-resistant cancer comprising phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient, and a method for diagnosing radiation-resistant cancer using the same. Further, the present invention relates to a biomarker composition for predicting the prognosis of radiation therapy for cancer patients including PMVK as an active ingredient, and a method for predicting the prognosis of radiation therapy for cancer patients using the composition.
암 치료 방법에는 외과적 수술, 화학적 약물치료, 방사선 치료 등이 있으며, 최근 들어 방사선 치료의 중요성이 더욱 커지고 있다. 외과적 수술이 어려운 경우, 방사선 치료만으로도 종양을 효율적으로 완치한 사례들이 보고되고 있고, 방사선을 이용한 종양 치료 방법도 해를 거듭하면서 발전하고 있어, 방사선 치료는 환자에게 특별한 고통이나 거부감 없이 체내의 종양을 효율적으로 치료할 수 있는 법으로 자리 잡고 있다. Cancer treatment methods include surgical operations, chemical medication, and radiotherapy. Radiation therapy is becoming more important in recent years. In cases where surgical treatment is difficult, radiation therapy alone has been effective in curing tumors. Radiation therapy has also been developed over the years, and radiation therapy has been applied to patients with no special pain or discomfort. As a way to treat it effectively.
그러나 암세포의 방사선 내성 획득, 고선량 방사선 치료 시 정상 조직의 손상 등이 방사선 치료의 효율을 저하시키는 문제점으로 지적되고 있어, 방사선 치료의 효율을　증진시키기　위한　방사선 치료 민감제에 대한　연구가　필요한 실정이다. 현재까지 보고된　방사선 치료 민감제는 주로 항암제들로서, 예를 들어 탁솔(taxol)과 시스플라틴(cisplatin)이 유방암, 자궁암. 폐암, 위암, 대장암 등의 고형암에서 방사선 치료 민감제로 사용될 수 있다고 보고된 바 있다. 그러나 이들방사선 치료 민감제들은 그 자체가 항암제로서 사용되는 물질이어서 높은 부작용을 나타내므로 그 이용이 제한적이라는 문제점이 있다.However, the acquisition of radiation tolerance of cancer cells and the damage of normal tissues during high-dose radiation therapy have been pointed out as a problem of decreasing the efficiency of radiation therapy. Therefore, it is necessary to study the radiation therapy sensitizer to improve the efficiency of radiation therapy . Radiation therapy sensitizers reported to date are mainly anticancer drugs, such as taxol and cisplatin, for breast, uterine, and cervical cancer. It has been reported that it can be used as a radiotherapy sensitizer in solid tumors such as lung cancer, stomach cancer and colon cancer. However, these radiation-sensitive sensitizers are substances that are used as anticancer drugs themselves, and thus exhibit high side effects, and thus their use is limited.
따라서, 이러한 기존 방사선 치료 민감제의 한계를 벗어나, 방사선 치료 결과를 예측할 수 있을 뿐만 아니라 방사선 저항성을 갖는 세포에 민감성을 증가시키기 위한 분자 신호에 대한 연구가 필요한 실정이다.Therefore, it is necessary to study the molecular signal to increase the sensitivity to radiation-resistant cells as well as to predict the outcome of radiation treatment beyond the limit of the conventional radiation therapy sensitizer.
본 발명의 목적은 PMVK를 유효성분으로 포함하는 방사선 저항성 암 진단용 바이오마커 조성물, PMVK의 발현 수준을 측정할 수 있는 제제를 유효성분으로 포함하는 방사선 저항성 암 진단용 조성물을 제공하는 것이다.It is an object of the present invention to provide a radiation-resistant cancer diagnostic biomarker composition containing PMVK as an active ingredient, and a composition capable of measuring the expression level of PMVK as an active ingredient.
본 발명의 다른 목적은 PMVK을 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 바이오마커 조성물, PMVK의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 조성물을 제공하고자 한다.Another object of the present invention is to provide a biomarker composition for predicting the prognosis of radiation therapy for a cancer patient comprising PMVK as an active ingredient, a composition for predicting the prognosis of radiation therapy for cancer patients, which comprises a preparation capable of measuring the expression level of PMVK .
본 발명의 또 다른 목적은 PMVK 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 암세포에 대한 방사선 민감성 증진용 약학 조성물을 제공하는 것이다.It is still another object of the present invention to provide a pharmaceutical composition for promoting radiation sensitivity to cancer cells comprising PMVK protein expression or activity inhibitor as an active ingredient.
본 발명의 또 다른 목적은 PMVK 단백질의 발현 및 활성 수준을 이용한 방사선 저항성 암 진단 방법 및 암 환자의 방사선 치료 예후 예측 방법을 제공하는 것이다.Another object of the present invention is to provide a method for diagnosing radiation-resistant cancer using PMVK protein expression and activity level and a method for predicting the prognosis of radiation therapy for cancer patients.
본 발명의 또 다른 목적은 PMVK 단백질의 발현 수준 측정을 통한 암세포에 대한 방사선 민감성 증진제 스크리닝 방법을 제공하는 것이다.It is another object of the present invention to provide a method for screening a radiation sensitivity enhancer for cancer cells by measuring the expression level of PMVK protein.
본 발명의 또 다른 목적은 방사선 저항성 암 진단을 위한 바이오마커로서, PMVK 단백질 또는 이를 코딩하는 유전자의 용도를 제공하는 것이다.It is still another object of the present invention to provide a biomarker for the diagnosis of radiation-resistant cancer, which uses the PMVK protein or a gene encoding the PMVK protein.
본 발명의 또 다른 목적은 방사선 저항성 암 진단을 위한 PMVK의 발현수준을 측정할 수 있는 제제의 용도를 제공하는 것이다.It is another object of the present invention to provide a use of an agent capable of measuring the level of expression of PMVK for the diagnosis of radiation resistant cancer.
본 발명의 또 다른 목적은 암 환자의 방사선 치료 예후 예측을 위한 바이오마커로서, PMVK 단백질 또는 이를 코딩하는 유전자의 용도를 제공하는 것이다.It is still another object of the present invention to provide the use of the PMVK protein or a gene encoding the PMVK protein as a biomarker for predicting the prognosis of radiation therapy in cancer patients.
본 발명의 또 다른 목적은 암 환자의 방사선 치료 예후 예측을 위한 PMVK의 발현수준을 측정할 수 있는 제제의 용도를 제공하는 것이다.It is another object of the present invention to provide a use of an agent capable of measuring the expression level of PMVK for predicting the prognosis of radiation therapy in a cancer patient.
본 발명의 또 다른 목적은 암세포에 대한 방사선 민감성 증진을 위한 약제를 제조하기 위한 PMVK 단백질 발현 또는 활성 억제제의 용도를 제공하는 것이다.It is a further object of the present invention to provide the use of PMVK protein expression or activity inhibitors for the manufacture of a medicament for enhancing radiation sensitivity to cancer cells.
상기 목적을 달성하기 위하여, 본 발명은 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 방사선 저항성 암 진단용 바이오마커 조성물을 제공한다.In order to achieve the above object, the present invention provides a biomarker composition for diagnosing radiation-resistant cancer, comprising as an active ingredient a phosphomevalonate kinase (PMVK) protein or a gene encoding the same.
또한, 본 발명은 PMVK의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 방사선 저항성 암 진단용 조성물 및 이를 포함하는 방사선 저항성 암 진단용 키트를 제공한다.The present invention also provides a composition for the diagnosis of radiation-resistant cancer, which comprises a preparation capable of measuring the expression level of PMVK as an active ingredient, and a radiation-resistant cancer diagnostic kit comprising the same.
상기 다른 목적을 달성하기 위하여, 본 발명은 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 바이오마커 조성물을 제공한다.In order to accomplish the above object, the present invention provides a biomarker composition for predicting the radiation therapy prognosis of a cancer patient, which comprises a phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient.
또한, 본 발명은 PMVK의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 조성물 및 이를 포함하는 암 환자의 방사선 치료 예후 예측용 키트를 제공한다.The present invention also provides a composition for predicting the prognosis of radiation therapy for cancer patients, which comprises a preparation capable of measuring the expression level of PMVK, and a kit for predicting the prognosis of radiation therapy for cancer patients containing the composition.
상기 또 다른 목적을 달성하기 위하여, 본 발명은 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 암세포에 대한 방사선 민감성 증진용 약학 조성물을 제공한다.According to another aspect of the present invention, there is provided a pharmaceutical composition for promoting radiation sensitivity to a cancer cell comprising, as an active ingredient, a phosphomevalonate kinase (PMVK) protein expression or activity inhibitor.
상기 또 다른 목적을 달성하기 위하여, 본 발명은 (1) 암 환자에서 분리된 시료로부터 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 측정하는 단계; (2) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 대조군 시료와 비교하는 단계; 및 (3) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준이 대조군 시료보다 높을 경우 방사선 저항성 암이라고 판단하는 단계를 포함하는 방사선 저항성 암 진단에 필요한 정보를 제공하는 방법을 제공한다.(1) measuring the mRNA expression level of PMVK gene or the expression level of PMVK protein from a sample isolated from a cancer patient; (2) comparing the mRNA expression level of the PMVK gene or the expression level of the PMVK protein with a control sample; And (3) determining that the mRNA expression level of the PMVK gene or the expression level of the PMVK protein is higher than that of the control sample, a step of determining that the cancer is a radiation-resistant cancer.
또한, 본 발명은 (1) 암 환자에서 분리된 시료로부터 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 측정하는 단계; (2) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 대조군 시료와 비교하는 단계; 및 (3) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준이 대조군 시료보다 높을 경우 방사선 치료 예후가 좋지 않다고 판단하는 단계를 포함하는 암 환자의 방사선 치료 예후 예측에 필요한 정보를 제공하는 방법을 제공한다.(1) measuring the mRNA expression level or PMVK protein expression level of a PMVK gene from a sample isolated from a cancer patient; (2) comparing the mRNA expression level of the PMVK gene or the expression level of the PMVK protein with a control sample; And (3) determining that the prognosis of radiotherapy is poor if the mRNA expression level of the PMVK gene or the expression level of the PMVK protein is higher than that of the control sample, to provide information necessary for predicting the prognosis of radiation therapy for cancer patients to provide.
상기 또 다른 목적을 달성하기 위하여, 본 발명은 (1) 암세포에 시험물질을 접촉시키는 단계; (2) 상기 시험물질을 접촉한 암세포에서 PMVK 단백질의 발현 또는 활성 정도를 측정하는 단계; 및 (3) 대조군 시료와 비교하여 상기 PMVK 단백질의 발현 또는 활성 정도가 감소한 시험물질을 선별하는 단계를 포함하는 암세포에 대한 방사선 민감성 증진제 스크리닝 방법을 제공한다.According to another aspect of the present invention, there is provided a method for detecting cancer, comprising the steps of: (1) contacting a test substance with a cancer cell; (2) measuring the expression or activity level of PMVK protein in the cancer cells in contact with the test substance; And (3) selecting a test substance whose expression or activity level of the PMVK protein is decreased as compared with a control sample. The present invention also provides a method for screening a radiation sensitivity enhancer for cancer cells.
또한, 본 발명은 방사선 저항성 암 진단을 위한 바이오마커로서, PMVK 단백질 또는 이를 코딩하는 유전자의 용도를 제공한다.The present invention also provides the use of the PMVK protein or a gene encoding the same as a biomarker for the diagnosis of radiation-resistant cancer.
또한, 본 발명은 방사선 저항성 암 진단을 위한 PMVK의 발현수준을 측정할 수 있는 제제의 용도를 제공한다.The present invention also provides the use of an agent capable of measuring the level of expression of PMVK for the diagnosis of radiation resistant cancer.
또한, 본 발명은 암 환자의 방사선 치료 예후 예측을 위한 바이오마커로서, PMVK 단백질 또는 이를 코딩하는 유전자의 용도를 제공한다.In addition, the present invention provides a biomarker for predicting the prognosis of radiation therapy in a cancer patient, which uses the PMVK protein or a gene encoding the PMVK protein.
또한, 본 발명은 암 환자의 방사선 치료 예후 예측을 위한 PMVK의 발현수준을 측정할 수 있는 제제의 용도를 제공한다.The present invention also provides the use of a pharmaceutical agent capable of measuring the level of expression of PMVK for predicting the prognosis of radiation therapy in cancer patients.
또한, 본 발명은 암세포에 대한 방사선 민감성 증진을 위한 약제를 제조하기 위한 PMVK 단백질 발현 또는 활성 억제제의 용도를 제공한다.The present invention also provides the use of a PMVK protein expression or activity inhibitor for the manufacture of a medicament for enhancing radiation sensitivity to cancer cells.
본 발명은 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 방사선 저항성 암 진단용 바이오마커 조성물 또는 방사선 치료 예후 예측용 바이오마커 조성물에 관한 것으로서, 사람 폐암 세포인 A549 세포에서 방사선의 민감도를 증진시킬 수 있는 PMVK 유전자를 규명하고, shRNA을 이용하여 A549 세포 및 췌장암 세포인 Miapaca-2 세포를 대상으로 안정 세포주를 구축한 후 세포와 동물 실험을 통하여 방사선 민감도 증진 효과를 검증하였으며, 더 나아가 임상적 데이터 분석을 통하여 PMVK의 과발현이 방사선의 저항성에 기여함을 확인하였는 바, 사람의 폐암 및 췌장암에서 PMVK를 방사선 저항성 극복을 위한 치료 표적 유전자로 활용할 수 있으므로, PMVK를 표적으로 방사선 민감도를 높이는 유전자 치료제 또는 기타 약물을 개발하여 방사선 치료 효과를 한층 증가시킬 수 있다.The present invention relates to a biomarker composition for diagnosing radiation-resistant cancer or a biomarker composition for predicting the prognosis of radiation therapy, which comprises a phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient, The PMVK gene, which can enhance the sensitivity of radiation in A549 cells, was identified, and A549 cells and Miapaca-2 cells of pancreatic cancer cells were constructed using shRNA, And PMVK overexpression contributes to the resistance of radiation through clinical data analysis. PMVK can be used as a therapeutic target gene to overcome radiation resistance in human lung and pancreatic cancer. Therefore, PMVK Genes that increase radiation sensitivity to targets Developed ryoje or other substances can be further increased the therapeutic effect of radiation.
도 1은 본 발명의 일 실시예에서, 인간 키나아제 siRNA 라이브러리 스크리닝을 통해 방사선 민감도 증진 유전자 PMVK를 확인한 결과를 나타낸 것이다(A: A549 인간 폐암 세포주에서 인간 키나아제 siRNA 라이브러리 스크리닝을 통해 방사선 민감도 증진 유전자 PMVK를 확인한 결과, B: PMVK siRNA에 의한 PMVK 단백질 발현 억제 및 방사선 처리시 세포 생존율 감소 결과를 확인한 결과임).FIG. 1 shows the result of confirming the radiation sensitivity-enhancing gene PMVK through human kinase siRNA library screening in one embodiment of the present invention. (A: A549 human lung cancer cell line was screened by human kinase siRNA library to determine the radiation sensitivity promoting gene PMVK As a result, B: PMVK siRNA inhibited the expression of PMVK protein and the result of reduction of cell viability was confirmed by radiation treatment).
도 2는 본 발명의 일 실시예에서, PMVK shRNA 안정 세포주를 구축하고, 구축된 세포에 방사선을 처리하여 집락 형성 생존 분석법을 시행한 결과를 나타낸 것이다(A: A549/shPMVK 안정 세포주에서 PMVK 단백질 발현 억제 효과를 확인한 결과, B: A549/shPMVK 안정 세포주에서 방사선이 집락 형성에 미치는 영향을 확인한 결과, C: Miapaca-2/shPMVK 안정 세포주에서 PMVK 단백질 발현 억제 효과를 확인한 결과, D: Miapaca-2/shPMVK 안정 세포주에서 방사선이 집락 형성에 미치는 영향을 확인한 결과임).FIG. 2 shows the result of performing colonization formation survival analysis by constructing a PMVK shRNA stable cell line and treating the established cells with radiation, in one embodiment of the present invention. (A: Expression of PMVK protein in A549 / shPMVK stable cell line The inhibitory effects of D: Miapaca-2 / shPMVK on the inhibition of PMVK protein expression in C: Miapaca-2 / shPMVK stable cell line were investigated as a result of confirming the effect of radiation on colony formation in B: A549 / shPMVK stable cell line. The effect of radiation on colony formation was confirmed in shPMVK stable cell lines.
도 3은 본 발명의 일 실시예에서, A549/shPMVK 세포 이식 마우스 모델에서의 방사선에 대한 효능 평가에 대한 결과를 나타낸 것이다(A: 암의 성장을 부피로 나타내 비교한 것, B: 마우스의 체중을 비교한 것, C: 적출된 암의 크기를 비교한 것, D: 적출된 암의 무게를 비교한 것임).Figure 3 shows the results of an evaluation of efficacy against radiation in an A549 / shPMVK cell transplantation mouse model in one embodiment of the present invention (A: volume growth of cancer compared to volume B: weight of mouse , C: comparison of the size of the extracted cancer, and D: comparison of the weight of the extracted cancer).
도 4는 본 발명의 일 실시예에서, 폐암 환자군에 방사선 처리 시 PMVK 발현에 따른 임상적 유전체 데이터 분석 결과를 나타낸 것이다.Figure 4 shows the results of clinical genomic data analysis according to the expression of PMVK in radiation treatment of lung cancer patients in one embodiment of the present invention.
도 5는 본 발명의 일 실시예에서, Miapaca-2/shPMVK 세포 이식 마우스 모델에서의 방사선에 대한 효능 평가에 대한 결과를 나타낸 것이다.Figure 5 shows the results of an efficacy assessment for radiation in a Miapaca-2 / shPMVK cell transplant mouse model in one embodiment of the invention.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 방사선 저항성 암 진단용 바이오마커 조성물을 제공한다.The present invention provides a biomarker composition for diagnosing radiation-resistant cancer, which comprises as an active ingredient a phosphomevalonate kinase (PMVK) protein or a gene coding therefor.
바람직하게는, 상기 바이오마커 조성물은 기존에 알려진 방사선 저항성 바이오마커를 추가적으로 더 포함할 수 있으며, 기존에 알려진 방사선 저항성 바이오마커는 CD133, CD144 및 CD24 등이 있으나, 이에 제한되는 것은 아니다.Preferably, the biomarker composition further comprises a known radiation resistance biomarker, and known radiation resistance biomarkers include, but are not limited to, CD133, CD144, and CD24.
본 명세서에서 용어 "진단"은 특정 질병 또는 질환에 대한 한 객체의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)를 판정하는 것, 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링하는 것)를 포함한다.The term " diagnosing " herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of the object, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).
또한, 본 발명은 PMVK의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 방사선 저항성 암 진단용 조성물을 제공한다.In addition, the present invention provides a radiation-resistant cancer diagnostic composition comprising as an active ingredient a preparation capable of measuring the expression level of PMVK.
상세하게는, 상기 PMVK의 발현 수준을 측정할 수 있는 제제는 상기 PMVK 유전자에 특이적으로 결합하는 프라이머 또는 프로브, 상기 PMVK 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머 또는 화합물일 수 있으나, 이에 제한되지는 않는다.In detail, the agent capable of measuring the expression level of PMVK may be a primer or a probe specifically binding to the PMVK gene, an antibody, a peptide, an aptamer or a compound specifically binding to the PMVK protein, But is not limited thereto.
상기 암은 폐암 또는 췌장암일 수 있으나, 이에 제한되지는 않는다.The cancer may be lung cancer or pancreatic cancer, but is not limited thereto.
또한, 본 발명은 상기 조성물을 포함하는 방사선 저항성 암 진단용 키트를 제공한다.The present invention also provides a radiation-resistant cancer diagnostic kit comprising the composition.
더불어, 본 발명은 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 바이오마커 조성물을 제공한다.In addition, the present invention provides a biomarker composition for predicting the radiotherapeutic prognosis of a cancer patient comprising a phosphomevalonate kinase (PMVK) protein or a gene coding therefor as an active ingredient.
본 명세서에서 용어 "예후 예측"은 질환의 경과 및 결과를 미리 예측하는 행위를 의미한다. 보다 구체적으로, 예후 예측이란 질환의 치료 후 경과는 환자의 생리적 또는 환경적 상태에 따라 달라질 수 있으며, 이러한 환자의 상태를 종합적으로 고려하여 치료 후 병의 경과를 예측하는 모든 행위를 의미하는 것으로 해석될 수 있다.As used herein, the term " prognostic prediction " refers to an act of predicting the course and outcome of a disease. More specifically, the prognosis prediction means that the progress of the disease after the treatment depends on the physiological or environmental condition of the patient, and it means all actions that predict the course of the disease after treatment considering the condition of the patient in a comprehensive manner .
본 발명의 목적상 상기 예후 예측은 암 환자의 방사선 치료 후, 질환의 경과및 완치 여부를 미리 예상하여 암 환자의 무병 생존율 또는 생존율을 예측하는 행위로 해석될 수 있다. 예를 들어, "예후가 좋다"라고 예측하는 것은 방사선 치료 후 암 환자의 무병 생존율 또는 생존율이 높은 수준을 나타내어, 암 환자가 치료될 가능성이 높다는 것을 의미하고, "예후가 나쁘다"라고 예측하는 것은 방사선 치료 후 암 환자의 무병 생존율 또는 생존율이 낮은 수준을 나타내어, 암 환자로부터 암이 재발하거나 또는 암으로 인하여 사망할 가능성이 높다는 것을 의미한다.For the purpose of the present invention, the prognosis prediction can be interpreted as predicting the disease-free survival rate or the survival rate of the cancer patient by anticipating the course of the disease and the cure after the radiation therapy of the cancer patient. For example, predicting a "good prognosis" indicates a high level of disease-free survival or survival rate in cancer patients after radiation therapy, which implies that cancer patients are more likely to be treated, and the prediction of "poor prognosis" The disease-free survival rate or survival rate of cancer patients after radiation therapy is low, meaning that cancer is likely to recur from cancer patients or die from cancer.
또한, 본 발명은 PMVK의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 조성물을 제공한다.The present invention also provides a composition for predicting the prognosis of radiation therapy for a cancer patient, which comprises a preparation capable of measuring the expression level of PMVK as an active ingredient.
이때, PMVK의 발현 수준을 측정할 수 있는 제제는 상기 PMVK 유전자에 특이적으로 결합하는 프라이머 또는 프로브, 상기 PMVK 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머 또는 화합물일 수 있다.Here, the agent capable of measuring the expression level of PMVK may be a primer or a probe specifically binding to the PMVK gene, an antibody, a peptide, an aptamer or a compound specifically binding to the PMVK protein.
상기 암은 폐암 또는 췌장암일 수 있으나, 이에 제한되지는 않는다.The cancer may be lung cancer or pancreatic cancer, but is not limited thereto.
또한, 본 발명은 상기 조성물을 포함하는 암 환자의 방사선 치료 예후 예측용 키트를 제공한다.In addition, the present invention provides a kit for predicting the prognosis of radiation therapy for a cancer patient comprising the composition.
본 명세서에서 용어 "프라이머"는 짧은 자유 3-말단 수산화기(free 3'-hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사을 위한 시작 지점으로서 작용하는 짧은 핵산 서열을 말한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(즉, DNA 폴리머라제 또는 역전사효소) 및 상이한 4가지의 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성을 개시할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 기술에 따라 적절히 선택될 수 있다.As used herein, the term " primer " refers to a nucleic acid sequence capable of forming base pairs with a complementary template with a short free 3'-hydroxyl group and having a short Refers to a nucleic acid sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.
본 명세서에서 용어 "프로브"는 mRNA외 특이적으로 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링되어 있어서 특정 mRNA의 존재 유무, 발현량을 확인할 수 있다. 프로브는 올리고뉴클레오타이드(oligonucleotide) 프로브, 단일가닥 DNA(single strand DNA) 프로브, 이중가닥 DNA(double strand DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적절한 프로브의 선택 및 혼성화 조건은 당해 기술 분야에 공지된 기술에 따라 적절히 선택할 수 있다.As used herein, the term " probe " means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to an mRNA. The presence or absence of a specific mRNA, Can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. Selection of suitable probes and hybridization conditions can be appropriately selected according to techniques known in the art.
본 명세서에서 용어 "항체"는 당해 기술분야에 공지된 용어로서 항원성 부위에 대하여 지시되는 특이적인 면역 글로불린을 의미한다. 본 발명에서의 항체는 본 발명의 PMVK에 대해 특이적으로 결합하는 항체를 의미하며, 당해 기술분야의 통상적인 방법에 따라 항체를 제조할 수 있다. 상기 항체의 형태는 폴리클로날 항체 또는 모노클로날 항체를 포함하며, 모든 면역글로불린 항체가 포함된다. 상기 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 갖는 완전한 형태를 의미한다. 또한, 상기 항체는 인간화 항체 등의 특수 항체도 포함된다.As used herein, the term " antibody " means a specific immunoglobulin as indicated in the art and directed against an antigenic site. An antibody in the present invention refers to an antibody that specifically binds to the PMVK of the present invention, and the antibody can be produced according to a conventional method in the art. The forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes a special antibody such as a humanized antibody.
본 명세서에서 용어 "펩타이드"는 표적 물질에 대한 결합력 높은 장점이 있으며, 열/화학 처리시에도 변성이 일어나지 않는다. 또한, 분자 크기가 작기 때문에 다른 단백질에 붙여서 융합 단백질로의 이용이 가능하다. 구체적으로 고분자 단백질 체인에 붙여서 이용이 가능하므로 진단키트 및 약물전달 물질로 이용될 수 있다.As used herein, the term " peptide " has a high binding capacity to the target material and does not cause denaturation during thermal / chemical treatment. In addition, since the molecular size is small, it can be used as a fusion protein by attaching to other proteins. It can be used as a diagnostic kit and a drug delivery material because it can be specifically attached to a polymer protein chain.
본 명세서에서 용어 "앱타머(aptamer)"란, 그 자체로 안정된 삼차 구조를 가지면서 표적 분자에 높은 친화성과 특이성으로 결합할 수 있는 특징을 가진 특별한 종류의 단일가닥 핵산(DNA, RNA 또는 변형핵산)으로 구성된 폴리뉴클레오티드의 일종을 의미한다. 상술한 바와 같이, 앱타머는 항체와 동일하게 항원성 물질에 특이적으로 결합할 수 있으면서도, 단백질보다 안정성이 높고, 구조가 간단하며, 합성이 용이한 폴리뉴클레오티드로 구성되어 있으므로, 항체를 대체하여 사용될 수 있다.The term " aptamer " as used herein refers to a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid having a stable tertiary structure by itself and having a characteristic capable of binding with high affinity and specificity to a target molecule ). ≪ / RTI > As described above, since the aptamer is composed of a polynucleotide which is capable of specifically binding to an antigenic substance like the antibody and is more stable than the protein, has a simple structure, and is easy to synthesize, .
또한, 본 발명의 키트는 마커 성분에 특이적으로 결합하는 항체, 기질과의 반응에 의해서 발색하는 표지체가 접합된 2차 항체 접합체(conjugate), 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 효소반응 정지용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있다.In addition, the kit of the present invention comprises an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a labeling substance that is colored by the reaction with the substrate, a coloring substrate solution that reacts with the labeling substance, Enzyme reaction termination solutions, and the like, and can be made from a number of separate packaging or compartments, including the reagent components used.
더불어, 본 발명은 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 암세포에 대한 방사선 민감성 증진용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for promoting radiation sensitivity to a cancer cell comprising, as an active ingredient, a phosphomevalonate kinase (PMVK) protein expression or activity inhibitor.
상세하게는, 상기 PMVK 발현 억제제는 PMVK 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, 작은 간섭 RNA(small interfering RNA; siRNA) 및 짧은 헤어핀 RNA(short hairpin RNA; shRNA)로 구성된 군으로부터 선택된 어느 하나일 수 있고, 상기 PMVK 활성 억제제는 PMVK 단백질에 특이적으로 결합하는 저분자 화합물, 펩티드, 펩티드 미메틱스, 앱타머, 항체 및 천연물로 구성된 군으로부터 선택된 어느 하나일 수 있으나, 이에 제한되는 것은 아니다.Specifically, the PMVK expression inhibitor may be any one selected from the group consisting of an antisense nucleotide complementarily binding to the mRNA of the PMVK gene, a small interfering RNA (siRNA) and a short hairpin RNA (shRNA) And the PMVK activity inhibitor may be any one selected from the group consisting of low molecular compounds, peptides, peptide mimetics, aptamers, antibodies and natural substances that specifically bind to the PMVK protein, but the present invention is not limited thereto.
이때, 상기 암세포는 폐암 세포 또는 췌장암 세포일 수 있다.At this time, the cancer cells may be lung cancer cells or pancreatic cancer cells.
본 발명의 약학 조성물은 화학물질, 뉴클레오타이드, 안티센스, siRNA 올리고뉴클레오타이드 및 천연물 추출물을 유효성분으로 포함할 수 있다. 본 발명의 약학 조성물 또는 복합 제제는 유효 성분 이외에 약제학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등의 가용화제를 사용할 수 있다. 본 발명의 약학 조성물은 투여를 위해서 유효 성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약학 조성물로 바람직하게 제제화할 수 있다. 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.The pharmaceutical composition of the present invention may contain a chemical substance, a nucleotide, an antisense, an siRNA oligonucleotide and a natural product extract as an active ingredient. The pharmaceutical composition or combination preparation of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants , A lubricant or a flavoring agent may be used. The pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
본 발명의 약학 조성물의 약제 제제 형태는 과립제, 산제, 피복정, 정제, 캡슐제, 좌제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 및 활성 화합물의 서방출형 제제 등이 될 수 있다. 본 발명의 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다. 본 발명의 약학 조성물의 유효성분의 유효량은 질환의 예방 또는 치료 시 요구되는 양을 의미한다. 따라서, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 이에 제한되는 것은 아니나, 예컨대, 성인의 경우, 1일 1회 내지 수회 투여시, 본 발명의 조성물은 1일 1회 내지 수회 투여 시, 화합물일 경우 0.1 ng/kg 내지 10 g/kg, 폴리펩타이드, 단백질 또는 항체일 경우 0.1 ng/kg 내지 10 g/kg, 안티센스 뉴클레오타이드, siRNA, shRNAi, miRNA일 경우 0.01 ng/kg 내지 10 g/kg의 용량으로 투여할 수 있다.Pharmaceutical dosage forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, suspending agents or injectable solutions or suspensions . The pharmaceutical compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, ≪ / RTI > An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, once to several times a day, the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg for a compound once to several times a day, , 0.1 ng / kg to 10 g / kg for a protein or an antibody, 0.01 ng / kg to 10 g / kg for an antisense nucleotide, siRNA, shRNAi or miRNA.
더욱이, 본 발명은 (1) 암 환자에서 분리된 시료로부터 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 측정하는 단계; (2) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 대조군 시료와 비교하는 단계; 및 (3) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준이 대조군 시료보다 높을 경우 방사선 저항성 암이라고 판단하는 단계를 포함하는 방사선 저항성 암 진단에 필요한 정보를 제공하는 방법을 제공한다.Further, the present invention provides a method for detecting cancer, comprising the steps of: (1) measuring the mRNA expression level or the PMVK protein expression level of a PMVK gene from a sample isolated from a cancer patient; (2) comparing the mRNA expression level of the PMVK gene or the expression level of the PMVK protein with a control sample; And (3) determining that the mRNA expression level of the PMVK gene or the expression level of the PMVK protein is higher than that of the control sample, a step of determining that the cancer is a radiation-resistant cancer.
또한, 본 발명은 (1) 암 환자에서 분리된 시료로부터 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 측정하는 단계; (2) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 대조군 시료와 비교하는 단계; 및 (3) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준이 대조군 시료보다 높을 경우 방사선 치료 예후가 좋지 않다고 판단하는 단계를 포함하는 암 환자의 방사선 치료 예후 예측에 필요한 정보를 제공하는 방법을 제공한다.(1) measuring the mRNA expression level or PMVK protein expression level of a PMVK gene from a sample isolated from a cancer patient; (2) comparing the mRNA expression level of the PMVK gene or the expression level of the PMVK protein with a control sample; And (3) determining that the prognosis of radiotherapy is poor if the mRNA expression level of the PMVK gene or the expression level of the PMVK protein is higher than that of the control sample, to provide information necessary for predicting the prognosis of radiation therapy for cancer patients to provide.
이때, 상기 암은 폐암 또는 췌장암일 수 있으나, 이에 제한되지는 않는다.At this time, the cancer may be lung cancer or pancreatic cancer, but is not limited thereto.
본 명세서에서 용어 "암 환자에서 분리된 시료"란 방사선 저항성 암 진단용 바이오 마커인 상기 PMVK 유전자 또는 PMVK 단백질의 발현 수준에 있어서, 대조군과 차이가 나는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액, 또는 뇨와 같은 시료를 포함하지만, 이에 한정되는 것은 아니다.As used herein, the term " a sample isolated from a cancer patient " refers to a level of expression of PMVK gene or PMVK protein, which is a biomarker for the diagnosis of radiation-resistant cancer, in tissues, cells, whole blood, serum, plasma, saliva, , A cerebrospinal fluid, or a sample such as urine.
상세하게는, 상기 mRNA 발현 수준을 측정하는 방법은 RT-PCR, 경쟁적 RT-PCR(Competitive RT-PCR), 실시간 RT-PCR (Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅 (Northern blotting) 및 DNA 칩을 이용하지만, 이에 한정되는 것은 아니다.In detail, the method for measuring the mRNA expression level may be RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) ), Northern blotting and DNA chips, but are not limited thereto.
상세하게는, 상기 단백질 발현 수준을 측정하는 방법은 웨스턴 블랏, ELISA(enzyme linked immunosorbent asay), 방사선면역분석(Radioimmunoassay; RIA), 방사면역확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케이트(rocket) 면역전기영동, 조직면역염색, 면역침전 분석법(Immunoprecipitation assay), 보체고정분석법 (Complement Fixation Assay), FACS 및 단백질 칩을 이용하지만, 이에 한정되는 것은 아니다.In detail, the method for measuring the protein expression level may be Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, But are not limited to, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, Complement Fixation Assays, FACS and protein chips.
본 명세서에서 "방사선 저항성 암 진단"은 암 환자의 방사선 치료 전략 및 방사선 치료 효과에 대한 예측을 위해 암세포가 방사선에 저항성인지 또는 민감성인지를 확인하기 위함이다.In the present specification, " radiation-resistant cancer diagnosis " is made to confirm whether cancer cells are resistant to radiation or sensitive to radiation therapy strategies and radiation therapy effects of cancer patients.
더불어, 본 발명은 (1) 암세포에 시험물질을 접촉시키는 단계; (2) 상기 시험물질을 접촉한 암세포에서 PMVK 단백질의 발현 또는 활성 정도를 측정하는 단계; 및 (3) 대조군 시료와 비교하여 상기 PMVK 단백질의 발현 또는 활성 정도가 감소한 시험물질을 선별하는 단계를 포함하는 암세포에 대한 방사선 민감성 증진제 스크리닝 방법을 제공한다.In addition, the present invention relates to (1) contacting a test substance to a cancer cell; (2) measuring the expression or activity level of PMVK protein in the cancer cells in contact with the test substance; And (3) selecting a test substance whose expression or activity level of the PMVK protein is decreased as compared with a control sample. The present invention also provides a method for screening a radiation sensitivity enhancer for cancer cells.
본 발명의 스크리닝 방법을 언급하면서 사용되는 용어 "시험물질"은 유전자의 발현량에 영향을 미치거나, 단백질의 발현 또는 활성에 영향을 미치는지 여부를 검사하기 위하여 스크리닝에서 이용되는 미지의 후보 물질을 의미한다. 상기 시료는 화학물질, 뉴클레오타이드, 안티센스-RNA, siRNA(small interference RNA) 및 천연물 추출물을 포함하나, 이에 제한되는 것은 아니다.The term " test substance " used in reference to the screening method of the present invention refers to an unknown candidate substance used in screening in order to examine whether it affects the expression amount of a gene or affects the expression or activity of a protein. do. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.
또한, 본 발명은 방사선 저항성 암 진단을 위한 바이오마커로서, PMVK 단백질 또는 이를 코딩하는 유전자의 용도를 제공한다.The present invention also provides the use of the PMVK protein or a gene encoding the same as a biomarker for the diagnosis of radiation-resistant cancer.
또한, 본 발명은 방사선 저항성 암 진단을 위한 PMVK의 발현수준을 측정할 수 있는 제제의 용도를 제공한다.The present invention also provides the use of an agent capable of measuring the level of expression of PMVK for the diagnosis of radiation resistant cancer.
또한, 본 발명은 암 환자의 방사선 치료 예후 예측을 위한 바이오마커로서, PMVK 단백질 또는 이를 코딩하는 유전자의 용도를 제공한다.In addition, the present invention provides a biomarker for predicting the prognosis of radiation therapy in a cancer patient, which uses the PMVK protein or a gene encoding the PMVK protein.
또한, 본 발명은 암 환자의 방사선 치료 예후 예측을 위한 PMVK의 발현수준을 측정할 수 있는 제제의 용도를 제공한다.The present invention also provides the use of a pharmaceutical agent capable of measuring the level of expression of PMVK for predicting the prognosis of radiation therapy in cancer patients.
또한, 본 발명은 암세포에 대한 방사선 민감성 증진을 위한 약제를 제조하기 위한 PMVK 단백질 발현 또는 활성 억제제의 용도를 제공한다.The present invention also provides the use of a PMVK protein expression or activity inhibitor for the manufacture of a medicament for enhancing radiation sensitivity to cancer cells.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.
<실시예 1> 인간 키나아제 siRNA 라이브러리(human kinases siRNA library) 스크리닝을 통한 방사선 민감도 증진 유전자 PMVK 확인<Example 1> Confirmation of PMVK gene promoting radiation sensitivity through screening of human kinases siRNA library
1. 실험 방법1. Experimental Method
A549 인간 폐암 세포(ATCC에서 구입)에서 다마콘(Dharmacon) siGENOME® SMARTpool® siRNA 라이브러리(718개 인간 단백질 키나아제: G-003505)를 이용하여 방사선에 대한 민감도를 증진시키는 유전자를 3차에 걸친 스크리닝을 통해 발굴하였다.A549 human lung cancer cells (purchased from ATCC) were subjected to a three-step screening of genes that enhance sensitivity to radiation using the Dharmacon siGENOME ® SMARTpool ® siRNA library (718 human protein kinase: G-003505) .
구체적으로, siRNA가 각각 50 nM 농도로 존재하는 96 웰 플레이트에 리포펙타민(Lipofectamine®) RNAiMAX 시약(Reagent)(Invitrogen: 13778-150) 0.5 ㎕을 첨가하였고, A549 세포를 0.3 × 104개로 시딩한 후 24시간 배양하였다. 배양된 세포에 6-MV 광자선 선형 가속기(6-MV photon beam linear accelerator)를 이용하여 8 Gy의 방사선을 처리하였다. 방사선 처리 후 9일째에 CCK-8 어세이를 통해 세포의 생존율을 3차에 걸쳐 분석하여, 그 결과, PMVK 유전자가 방사선에 대한 민감도와 관련이 있는 후보 유전자라는 정보를 획득하였다. 또한, siRNA 라이브러리에 존재하는 다마콘 siGENOME® SMARTpool® 인간 PMVK siRNA(표적 시퀀스(Target Sequences): UUUAUCCGCUCCAGACUUU(서열번호 1), CGAGAACCAUGGAGUUGAA(서열번호 2), AAUGUGGCCUGGACAACUU(서열번호 3), GGUGAGUGACACACGGAGA(서열번호 4); Cat# M-006782-01)에 의한 단백질의 넉-다운(knock-down) 여부는, PMVK siRNA가 50 nM 농도로 존재하는 24 웰 플레이트에 리포펙타민　(Lipofectamine®) RNAiMAX 시약(Reagent)(Invitrogen: 13778-150) 1 ㎕을 첨가하고, A549 세포를 3 × 104개로 시딩한 후 24시간 배양한 다음 하나의 웰에서 단백질을 추출한 뒤 PMVK 항체를 이용한 웨스턴 블롯팅을 수행함으로써 확인하였다. 남은 24 웰에는 방사선을 5 Gy, 10 Gy 처리한 후 CCK-8 어세이를 수행하여 세포의 생존율을 재확인하였다.Specifically, siRNA is Lipofectamine (Lipofectamine ®) RNAiMAX reagent (Reagent) in a 96-well plate, each present in a 50 nM concentration was added to 0.5 ㎕ (Invitrogen 13778-150), A549 cells seeded 0.3 × 10 4 pieces And cultured for 24 hours. The cultured cells were treated with 8 Gy of radiation using a 6-MV photon beam linear accelerator. On the 9th day after radiation treatment, cell viability was analyzed three times through the CCK-8 assay. As a result, information was obtained that the PMVK gene is a candidate gene related to the sensitivity to radiation. The siRNA library siGENOME ® SMARTpool ® human PMVK siRNA (Target Sequences: UUUAUCCGCUCCAGACUUU (SEQ ID NO: 1), CGAGAACCAUGGAGUUGAA (SEQ ID NO: 2), AAUGUGGCCUGGACAACUU (SEQ ID NO: 3), GGUGAGUGACACACGGAGA The knock-down of the protein by Cat # M-006782-01 was determined by incubating the 24-well plate in which PMVK siRNA was present at a concentration of 50 nM with Lipofectamine 　 (Lipofectamine ®) RNAiMAX reagent (Reagent) (Invitrogen: 13778-150) for 1 ㎕ added and the the A549 cell culture 24 hours after a 3 × 10 seeded into four, and then extracting the protein from one well behind using antibodies PMVK Western blotting. The remaining 24 wells were irradiated with 5 Gy and 10 Gy, and CCK-8 assay was performed to confirm the survival rate of the cells.
2. 실험 결과2. Experimental results
그 결과, 도 1A에 나타난 바와 같이, PMVK siRNA를 트랜스펙션(transfection)한 후 방사선을 처리했을 때, siScramble(대조군, 표적 시퀀스(Target Sequences): UGGUUUACAUGUCGACUAA(서열번호 5), UGGUUUACAUGUUGUGUGA(서열번호 6), UGGUUUACAUGUUUUCUGA(서열번호 7), UGGUUUACAUGUUUUCCUA(서열번호 8); 다마콘, 비-표적 풀(Non-Targeting pool), Cat# D-001810-10-20)에 방사선을 처리했을 때보다 세포의 생존율이 현저히 감소하는 것을 관찰할 수 있었다.As a result, when the PMVK siRNA was transfected and then treated with radiation, siScramble (control group, Target Sequences: UGGUUUACAUGUCGACUAA (SEQ ID NO: 5), UGGUUUACAUGUUGUGUGA (SEQ ID NO: 6 ), UGGUUUACAUGUUUUCUGA (SEQ ID NO: 7), UGGUUUACAUGUUUCCUA (SEQ ID NO: 8), Damacon, Non-Targeting pool, Cat # D-001810-10-20) As shown in Fig.
또한, 도 1B에 나타난 바와 같이, 웨스턴 블롯팅 수행 결과, PMVK siRNA에 의해 단백질이 넉-다운된 것을 확인하였고, 이때 방사선 처리 시에 세포의 생존율이 감소하는 것을 다시 한 번 확인하였다.Further, as shown in FIG. 1B, it was confirmed by Western blotting that the protein was knocked down by PMVK siRNA, and it was once again confirmed that the survival rate of the cells decreased during the radiation treatment.
따라서, 이러한 결과를 토대로 PMVK 발현의 감소가 방사선의 민감도 증진에 관여할 것으로 판단하였다.Based on these results, it was concluded that the reduction of PMVK expression would be involved in the enhancement of radiation sensitivity.
<실시예 2> PMVK shRNA 안정 세포주 구축 및 집락 형성 생존분석<Example 2> Construction of PMVK shRNA stable cell line and analysis of colony formation survival
1. 실험 방법1. Experimental Method
안정적인 PMVK의 넉-다운을 위해 PMVK shRNA 안정(stable) 세포주를 A549(폐암), Miapaca-2(췌장암; ATCC에서 구입) 세포에 구축하였다. 구체적으로, 다마콘 PMVK shRNA(Clone ID: V3LHS_350080, 성숙 안티센스(Mature Antisense): TCTGACTCAGCATCGTCCA; 서열번호 9)를 A549 세포와 Miapaca-2 세포에 FuGENE® 트랜스펙션 시약(Tansfection Reagent)(Roche: REF 04 709 713 001)을 사용하여 트랜스펙션 시킨 후, 퓨로마이신 4 ㎍/ml로 15일간 선택하여 A549/shPMVK 세포(2개 클론; A549/shPMVK#1 및 A549/shPMVK#3)와 Miapaca-2/shPMVK 세포주(3개 클론; Mia/shPMVK#1, Mia/shPMVK#2, Mia/shPMVK#3)를 구축하였고, 웨스턴 블롯팅을 통해 PMVK 단백질의 발현량을 확인하였다. PMVK shRNA stable cell lines were constructed on A549 (lung cancer) and Miapaca-2 (pancreatic cancer; purchased from ATCC) cells for stable knockdown of PMVK. Specifically, the damask cone PMVK shRNA (Clone ID: V3LHS_350080, mature antisense (Mature Antisense): TCTGACTCAGCATCGTCCA; SEQ ID NO: 9) (Roche FuGENE ® transfection reagent (Tansfection Reagent) to A549 cells and Miapaca-2 cells: REF 04 (Two clones: A549 / shPMVK # 1 and A549 / shPMVK # 3) and Miapaca-2 / shPMVK # 3 were selected for 15 days with puromycin 4 μg / ml for transfection, The shPMVK cell line (3 clones; Mia / shPMVK # 1, Mia / shPMVK # 2, Mia / shPMVK # 3) was constructed and the expression amount of PMVK protein was confirmed by Western blotting.
또한, 이렇게 구축된 A549/shPMVK 안정 세포주와 Miapaca-2/shPMVK 안정 세포주가 방사선의 민감도를 증진시키는지를 알아보기 위해, 두 세포주에 방사선을 처리한 후, 세포 집락 형성 생존분석법(colony forming assay)을 수행하였다. 즉, 6 웰 플레이트에 세포를 200개 또는 2000개씩 시딩한 후 방사선을 0, 2, 4, 5, 8, 10 Gy로 처리하였고, 7 내지 14일 뒤 20% 메탄올과 0.5% 크리스탈 바이올렛으로 콜로니를 염색하여 개수를 카운팅함으로써 정량적으로 분석하였다.In order to examine whether the thus constructed A549 / shPMVK stable cell line and Miapaca-2 / shPMVK stable cell line enhance the sensitivity of radiation, two cell lines were treated with radiation and then subjected to a colony forming assay Respectively. The cells were treated with 0, 2, 4, 5, 8, and 10 Gy of radiation after seeding 200 or 2000 cells in a 6-well plate. After 7 to 14 days, the cells were treated with 20% methanol and 0.5% And quantitatively analyzed by counting the number of staining.
2. 실험 결과2. Experimental results
그 결과, 도 2A 및 2C에 나타난 바와 같이, 친세포(parent; A549, Miapaca-2)와 비-침묵 대조군(Non-silencing control; A549/NS, Miapaca-2/NS)에 비교했을 때, 새로 구축된 A549/shPMVK 안정 세포주와 Miapaca-2/shPMVK 안정 세포주에서 PMVK 단백질 발현이 현저히 감소되었음을 확인하였다.As a result, compared to the parent cells (parent A549, Miapaca-2) and the non-silencing control (A549 / NS, Miapaca-2 / NS) PMVK protein expression was significantly reduced in the established A549 / shPMVK stable cell line and Miapaca-2 / shPMVK stable cell line.
또한, 도 2B 및 2D에 나타난 바와 같이, 이렇게 구축된 A549/shPMVK 안정 세포주와 Miapaca-2/shPMVK 안정 세포주를 대상으로 한 세포 집락 형성 생존분석법 결과, 집락 형성이 현저히 감소되는 것을 알 수 있었다.In addition, as shown in Figs. 2B and 2D, colonization formation was significantly reduced as a result of the cell colonization formation survival analysis of the thus constructed A549 / shPMVK stable cell line and Miapaca-2 / shPMVK stable cell line.
<< 실시예Example 3> A549/ 3> A549 / shPMVKshPMVK 세포 이식 마우스 모델에서 방사선에 대한 효능 평가 Evaluation of efficacy for radiation in cell transplantation mouse models
1. 실험 방법1. Experimental Method
생체 내(in vivo) 실험을 위해 누드 마우스 오른쪽 종아리에 A549, A549/NS, A549/shPMVK#1(서열번호 9의 shRNA로 트랜스펙션된 안정 A549 세포주) 및 A549/shPMVK#3(서열번호 9의 shRNA로 트랜스펙션된 안정 A549 세포주) 세포 1 × 106개를 각각 이식하여 암 조직을 형성시켰다. 이후 방사선 5 Gy를 이틀 간격으로 2번 조사하고 일주일에 3번 캘리퍼를 이용하여 크기를 측정하여 30일간 암 성장의 변화를 관찰하였고, 동시에 마우스 몸무게 또한 측정한 뒤, 30일일 때 암 조직을 적출하여 크기와 무게를 확인하였다. 이때, 암 크기는 하기 <식 1>에 기재된 대로 계산하였다.A549 / A549 / NS, A549 / shPMVK # 1 (stable A549 cell line transfected with shRNA of SEQ ID NO: 9) and A549 / shPMVK # 3 (SEQ ID NO: 9) in the nude right calf for in vivo experiments, the shRNA in a stable 1 × 10 6 A549 cell line) cells transfected dogs were each implanted to form a cancerous tissue. After irradiation, 5 Gy of radiation was irradiated twice at intervals of two days, and the size was measured using a caliper three times a week. The change of cancer growth was observed for 30 days. At the same time, the mouse body weight was also measured. Size and weight. At this time, the arm size was calculated as shown in the following formula (1).
[식 1][Formula 1]
암 크기(Tumor volume) = [세로(length) × 가로(width)2] × 0.5Tumor volume = [length × width 2 ] × 0.5
2. 실험 결과2. Experimental results
그 결과, 도 3에 나타난 바와 같이, A549/shPMVK#1과 #3에 방사선을 처리하였을 때, A549 또는 A549/NS에 방사선을 처리한 것보다 현저히 암 성장을 저해하고 있음을 확인하였고(도 3A 및 3C), 이때 마우스의 체중 변화는 보이지 않았다(도 3B). 실험 종료 시점에서 모든 마우스의 암 조직을 적출하여 암 무게를 측정하였을 때도 A549/shPMVK#1과 #3에 방사선을 처리한 암 조직의 무게가 가장 적음을 확인하였다(도 3D).As a result, as shown in FIG. 3, when A549 / shPMVK # 1 and # 3 were treated with radiation, it was confirmed that A549 or A549 / NS significantly inhibited tumor growth than radiation treatment (FIG. 3A And 3C), with no change in body weight of the mice (FIG. 3B). At the end of the experiment, the cancer tissues of all mice were measured to determine the weight of the cancer, and the weight of cancer tissues treated with radiation to A549 / shPMVK # 1 and # 3 was the lowest (FIG. 3D).
<실시예 4> 임상적 유전체 데이터 분석Example 4: Analysis of clinical genomic data
1. 실험방법1. Experimental Method
PMVK 발현과 환자에서 생존율과의 연관성을 조사하기 위하여, 기존에 보고된 폐암 환자군 유전체 데이터(GSE68465; NCBI. GEO, n=442) 분석을 통해 생존률을 조사하였다. 방사선 처리 환자군(n=65)과 그렇지 않은 환자군(n=364)으로 각각 나눈 후 환자 생존율을 PMVK의 발현에 따라 조사하였다.To investigate the association between PMVK expression and survival in patients, survival rates were examined by analysis of previously reported lung cancer genome data (GSE68465; NCBI.Geo, n = 442). Radiation - treated patients (n = 65) and non - treated patients (n = 364) were divided into two groups according to the expression of PMVK.
2. 실험결과2. Experimental results
그 결과, 도 4에 나타난 바와 같이, 방사선 처리 환자군에서는 PMVK의 발현이 높은 환자군일수록 생존율이 불량한 반면, PMVK의 발현이 낮은 환자군에 방사선 치료한 결과 환자의 생존율이 향상됨을 알 수 있었다. 그러나 방사선 치료를 하지 않은 환자군의 경우, PMVK의 발현 유무와 상관없이 생존율에 영향을 주지 않음을 확인하였다. As a result, as shown in FIG. 4, the survival rate of patients with high PMVK expression was poor in the radiotherapy patient group, while the survival rate of patients with low PMVK expression was improved by radiotherapy. However, the survival rate was not affected regardless of the presence or absence of PMVK in patients without radiation therapy.
이러한 결과들을 통해 PMVK의 발현과 방사선 치료 시 환자의 생존율이 밀접한 관계가 있고, 또한 PMVK의 억제를 통하여 방사선 치료 시 환자의 생존율 향상에 도움을 줄 수 있음을 확인하였다.These results indicate that PMVK expression is closely related to the survival rate of patients with radiotherapy, and that PMVK inhibition can improve patient survival in radiotherapy.
<< 실시예Example 5>  5> MiapacaMiapaca -2/-2/ shPMVKshPMVK 세포 이식 마우스 모델에서 방사선에 대한 효능 평가 Evaluation of efficacy for radiation in cell transplantation mouse models
1. 실험 방법1. Experimental Method
생체 내(in vivo) 실험을 위해 누드 마우스 오른쪽 종아리에 Miapaca-2, Miapaca-2/NS, Miapaca-2/shPMVK#2(서열번호 9의 shRNA로 트랜스펙션된 안정 Miapaca-2 세포주) 및 Miapaca-2/shPMVK#3(서열번호 9의 shRNA로 트랜스펙션된 안정 Miapaca-2 세포주) 세포 2 × 106개를 각각 이식하여 암 조직을 형성시켰다. 이후 방사선 2 Gy를 이틀 간격으로 3번 조사하고 일주일에 3번 캘리퍼를 이용하여 크기를 측정하여 30일간 암 성장의 변화를 관찰하였고, 동시에 마우스 몸무게 또한 측정하여 확인하였다. 이때, 암 크기는 하기 <식 1>에 기재된 대로 계산하였다.Miapaca-2 / NS, Miapaca-2 / shPMVK # 2 (stable Miapaca-2 cell line transfected with shRNA of SEQ ID NO: 9) and Miapaca-2 / shPMVK # 2 in the nude right calf for in vivo experiments, -2 / shPMVK # 3 and transplanted (transfected stable cell line Miapaca-2 shRNA in the specifications of SEQ ID NO: 9) cells 2 × 10 6 gae respectively to form a cancerous tissue. The radiation 2 Gy was irradiated three times at intervals of two days, and the size was measured using a caliper three times a week to observe the change of cancer growth for 30 days. At the same time, mouse weight was also measured and measured. At this time, the arm size was calculated as shown in the following formula (1).
[식 1][Formula 1]
암 크기(Tumor volume) = [세로(length) × 가로(width)2] × 0.5Tumor volume = [length × width 2 ] × 0.5
2. 실험 결과2. Experimental results
그 결과, 도 5에 나타난 바와 같이, Miapaca-2/shPMVK#2과 #3에 방사선을 처리하였을 때, Miapaca-2 또는 Miapaca-2/NS에 방사선을 처리한 것보다 현저히 암 성장을 저해하고 있음을 확인하였고(도 5A), 이때 마우스의 체중 변화는 보이지 않았다(도 5B).As a result, when irradiated with Miapaca-2 / shPMVK # 2 and # 3, Miapaca-2 or Miapaca-2 / NS significantly inhibited cancer growth as compared with the treatment with radiation (Fig. 5A), and no change in the weight of the mice was observed at this time (Fig. 5B).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. Do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (24)

  1. 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 방사선 저항성 암 진단용 바이오마커 조성물.A biomarker composition for diagnosing radiation-resistant cancer, comprising as an active ingredient a phosphomevalonate kinase (PMVK) protein or a gene encoding the same.
  2. 제 1 항에 있어서, 상기 암은 폐암 또는 췌장암인 것을 특징으로 하는 방사선 저항성 암 진단용 바이오마커 조성물.The biomarker composition for diagnosing radiation-resistant cancer according to claim 1, wherein the cancer is lung cancer or pancreatic cancer.
  3. PMVK의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 방사선 저항성 암 진단용 조성물.A composition for diagnosing radiation-resistant cancer, comprising as an active ingredient a preparation capable of measuring the expression level of PMVK.
  4. 제 3 항에 있어서, 상기 PMVK의 발현 수준을 측정할 수 있는 제제는 상기PMVK 유전자에 특이적으로 결합하는 프라이머 또는 프로브, 상기 PMVK 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머 또는 화합물인 것을 특징으로 하는 방사선 저항성 암 진단용 조성물.4. The method according to claim 3, wherein the agent capable of measuring the expression level of PMVK is a primer or a probe specifically binding to the PMVK gene, an antibody, a peptide, an aptamer or a compound specifically binding to the PMVK protein Wherein said radiation-resistant cancer diagnostic composition is a composition for diagnosing radiation-resistant cancer.
  5. 제 3 항에 있어서, 상기 암은 폐암 또는 췌장암인 것을 특징으로 하는 방사선 저항성 암 진단용 조성물.The composition for diagnosing radiation-resistant cancer according to claim 3, wherein the cancer is lung cancer or pancreatic cancer.
  6. 제 3 항 내지 제 5 항 중 어느 한 항의 조성물을 포함하는 방사선 저항성 암 진단용 키트.A radiation-resistant cancer diagnostic kit comprising the composition of any one of claims 3 to 5.
  7. 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 바이오마커 조성물.A biomarker composition for predicting the prognosis of radiation therapy in cancer patients comprising phosphomevalonate kinase (PMVK) protein or a gene encoding the same as an active ingredient.
  8. 제 7 항에 있어서, 상기 암은 폐암 또는 췌장암인 것을 특징으로 하는 암 환자의 방사선 치료 예후 예측용 바이오마커 조성물.8. The biomarker composition according to claim 7, wherein the cancer is lung cancer or pancreatic cancer.
  9. PMVK의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 암 환자의 방사선 치료 예후 예측용 조성물.A composition for predicting the prognosis of radiation therapy in a cancer patient, which comprises as an active ingredient a preparation capable of measuring the expression level of PMVK.
  10. 제 9 항에 있어서, 상기 PMVK의 발현 수준을 측정할 수 있는 제제는 상기PMVK 유전자에 특이적으로 결합하는 프라이머 또는 프로브, 상기 PMVK 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머 또는 화합물인 것을 특징으로 하는 암 환자의 방사선 치료 예후 예측용 조성물.10. The method according to claim 9, wherein the agent capable of measuring the expression level of PMVK is a primer or a probe specifically binding to the PMVK gene, an antibody, a peptide, an aptamer or a compound specifically binding to the PMVK protein A composition for predicting the prognosis of radiation therapy in a cancer patient.
  11. 제 9 항에 있어서, 상기 암은 폐암 또는 췌장암인 것을 특징으로 하는 암 환자의 방사선 치료 예후 예측용 조성물.10. The composition according to claim 9, wherein the cancer is lung cancer or pancreatic cancer.
  12. 제 9 항 내지 제 11 항 중 어느 한 항의 조성물을 포함하는 암 환자의 방사선 치료 예후 예측용 키트.A kit for predicting the prognosis of radiation therapy for cancer patients comprising the composition of any one of claims 9 to 11.
  13. 포스포메발로네이트 키나아제(phosphomevalonate kinase; PMVK) 단백질 발현 또는 활성 억제제를 유효성분으로 포함하는 암세포에 대한 방사선 민감성 증진용 약학 조성물.A pharmaceutical composition for enhancing radiation sensitivity to cancer cells comprising as an active ingredient phosphomevalonate kinase (PMVK) protein expression or activity inhibitor.
  14. 제 13 항에 있어서, 상기 PMVK 발현 억제제는 PMVK 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오타이드, 작은 간섭 RNA(small interfering RNA; siRNA) 및 짧은 헤어핀 RNA(short hairpin RNA; shRNA)로 구성된 군으로부터 선택된 어느 하나인 것을 특징으로 하는 암세포에 대한 방사선 민감성 증진용 약학 조성물.14. The method according to claim 13, wherein the PMVK expression inhibitor is selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA) and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the PMVK gene Or a pharmaceutically acceptable salt thereof.
  15. 제 13 항에 있어서, 상기 PMVK 활성 억제제는 PMVK 단백질에 특이적으로 결합하는 저분자 화합물, 펩티드, 펩티드 미메틱스, 앱타머, 항체 및 천연물로 구성된 군으로부터 선택된 어느 하나인 것을 특징으로 하는 암세포에 대한 방사선 민감성 증진용 약학 조성물.14. The method according to claim 13, wherein the PMVK activity inhibitor is any one selected from the group consisting of low molecular compounds, peptides, peptide mimetics, aptamers, antibodies, and natural substances that specifically bind to the PMVK protein A pharmaceutical composition for promoting radiation sensitivity.
  16. 제 13 항 내지 제 15 항 중 어느 한 항에 있어서, 상기 암세포는 폐암 세포 또는 췌장암 세포인 것을 특징으로 하는 암세포에 대한 방사선 민감성 증진용 약학 조성물.The pharmaceutical composition according to any one of claims 13 to 15, wherein the cancer cells are lung cancer cells or pancreatic cancer cells.
  17. (1) 암 환자에서 분리된 시료로부터 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 측정하는 단계;(1) measuring the mRNA expression level of the PMVK gene or the expression level of PMVK protein from a sample isolated from a cancer patient;
    (2) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 대조군 시료와 비교하는 단계; 및(2) comparing the mRNA expression level of the PMVK gene or the expression level of the PMVK protein with a control sample; And
    (3) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준이 대조군 시료보다 높을 경우 방사선 저항성 암이라고 판단하는 단계를 포함하는 방사선 저항성 암 진단에 필요한 정보를 제공하는 방법.(3) determining that the mRNA expression level of the PMVK gene or the expression level of the PMVK protein is higher than that of the control sample, and determining that the expression level of the PMVK protein is a radiation resistant cancer.
  18. (1) 암 환자에서 분리된 시료로부터 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 측정하는 단계;(1) measuring the mRNA expression level of the PMVK gene or the expression level of PMVK protein from a sample isolated from a cancer patient;
    (2) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준을 대조군 시료와 비교하는 단계; 및(2) comparing the mRNA expression level of the PMVK gene or the expression level of the PMVK protein with a control sample; And
    (3) 상기 PMVK 유전자의 mRNA 발현 수준 또는 PMVK 단백질의 발현 수준이 대조군 시료보다 높을 경우 방사선 치료 예후가 좋지 않다고 판단하는 단계를 포함하는 암 환자의 방사선 치료 예후 예측에 필요한 정보를 제공하는 방법.(3) A method for predicting the prognosis of radiation therapy for a cancer patient, comprising the step of determining that the prognosis of radiotherapy is poor when the level of mRNA expression of the PMVK gene or the level of PMVK protein expression is higher than that of the control sample.
  19. (1) 암세포에 시험물질을 접촉시키는 단계;(1) contacting a test substance with a cancer cell;
    (2) 상기 시험물질을 접촉한 암세포에서 PMVK 단백질의 발현 또는 활성 정도를 측정하는 단계; 및(2) measuring the expression or activity level of PMVK protein in the cancer cells in contact with the test substance; And
    (3) 대조군 시료와 비교하여 상기 PMVK 단백질의 발현 또는 활성 정도가 감소한 시험물질을 선별하는 단계를 포함하는 암세포에 대한 방사선 민감성 증진제 스크리닝 방법.(3) selecting a test substance whose expression or activity level of the PMVK protein is decreased as compared with a control sample.
  20. 방사선 저항성 암 진단을 위한 바이오마커로서, PMVK 단백질 또는 이를 코딩하는 유전자의 용도.A biomarker for the diagnosis of radiation-resistant cancer, which uses the PMVK protein or a gene coding therefor.
  21. 방사선 저항성 암 진단을 위한 PMVK의 발현수준을 측정할 수 있는 제제의 용도.The use of a preparation which is capable of measuring the expression level of PMVK for the diagnosis of radiation resistant cancer.
  22. 암 환자의 방사선 치료 예후 예측을 위한 바이오마커로서, PMVK 단백질 또는 이를 코딩하는 유전자의 용도.Use of a PMVK protein or a gene encoding the same as a biomarker for predicting the prognosis of radiation therapy in a cancer patient.
  23. 암 환자의 방사선 치료 예후 예측을 위한 PMVK의 발현수준을 측정할 수 있는 제제의 용도.Use of a preparation capable of measuring the expression level of PMVK for predicting radiation therapy prognosis in cancer patients.
  24. 암세포에 대한 방사선 민감성 증진을 위한 약제를 제조하기 위한 PMVK 단백질 발현 또는 활성 억제제의 용도.Use of PMVK protein expression or activity inhibitor for the manufacture of a medicament for enhancing radiation sensitivity to cancer cells.
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